Transthyretin's Regulatory Role in Beta-Amyloid Aggregation and Toxicity Alzheimer's disease (AD) has been linked to deposition of beta-amyloid (A[unreadable]) as amyloid plaques in the brain. Transgenic mice expressing the human A[unreadable] precursor protein (APP) produce high levels of A[unreadable] and develop amyloid plaques, but they do not suffer the extensive neuronal cell death that is characteristic of AD. Recent studies have uncovered a possible explanation: these transgenic mice greatly increase the synthesis of the transport protein transthyretin (TTR), and TTR appears to protect the mice from the neurotoxic effects of A[unreadable]. The long-term goals of this project are to answer three questions that arise from these intriguing results: (1) how does TTR exert its protective activity? (2) why does this natural protective activity fail in AD? (3) can it be restored? Our hypothesis is that subtle changes in TTR tertiary and/or quaternary structure strongly modulate TTR- A[unreadable] interactions. In specific aim 1, several TTR mutants that differ in their tertiary and/or quaternary structure and stability will be produced and characterized. Each mutant will be screened for its ability to interfere with A[unreadable] aggregation, and to inhibit A[unreadable] toxicity in an in vitro cell culture model. The data will be analyzed to identify correlations between TTR structure and stability with its ability to alter A[unreadable] aggregation and toxicity. In specific aim 2, a detailed examination of the interaction between TTR (both wildtype and selected mutants) and A[unreadable] will be undertaken. Biophysical and biochemical tools such as circular dichroism, fluorescence, static and dynamic light scattering, crosslinking, and kinetic modeling will be employed. From these data will emerge molecular-level mechanistic insights into the nature of TTR-A[unreadable] association and the means by which TTR affects A[unreadable] aggregation kinetics. The goal of specific aim 3 is to identify small ligands that stabilize TTR and determine their influence on TTR's ability to modulate A[unreadable] aggregation and toxicity. In aim 4, TTR (wildtype and mutants) along with TTR-binding ligands will be tested for protection against A[unreadable] toxicity in ex vivo and in vivo mouse models. This will be achieved by using a newly developed assay in which stereotactic injection of A[unreadable] into mouse brain leads to loss of CA1 and dentate gyrus neurons, and by infection of astrocytes with adenovirus-TTR constructs. The project spans from characterization of the structure and stability of TTR (and mutants), through in vitro assessment of TTR's effect on A[unreadable] aggregation and toxicity, to in vivo evaluation of TTR efficacy at preventing neuronal cell death. These studies will provide a sound and rational basis for developing novel pharmacological approaches to preventing AD by enhancement of the natural defenses provided by TTR.